The present invention relates to methods and products thereof for expanding the host range of certain viruses, and more particularly to the expansion of a baculovirus' host range through the use of a promoter which is active in the target host. Viruses with expanded host range may be used to express exogenous genes in the target host and more specifically to produce polypeptides which are toxic to the host.
Interest in microbial insecticides has arisen as a result of the problems associated with the use of chemical pesticides. Chemical pesticides generally affect beneficial as well as non-beneficial species, and insects tend to acquire resistance to such chemicals so that new insect populations are resistant to the chemical pesticides. Moreover, pressures are brought to bear by environmentalists concerned about the effect of chemical pesticides on wildlife, by pest-control experts concerned about the effects of these pesticides on parasites and predators of the pests, by consumers concerned about toxic residues in food, and by public health officials concerned about human poisoning. Furthermore, research, development and production costs for chemical pesticides have soared, making them expensive in the developed nations, while in the developing nations, if pesticides are used at all, farmers select the least expensive, which are usually the most toxic.
Microorganisms (entomopathogens) present an opportunity for an alternative means of pest control and can play a role in integrated pest management thus reducing the dependence on chemical pesticides. Naturally occurring microorganisms or microbial by-products have been identified and found to be useful as insecticidal agents. A number of entomopathogens have relatively narrow host ranges, which has made it possible to reduce specific pest populations while natural predators and beneficial insects are preserved or given the opportunity to become reestablished. Entomopathogens which are useful for insect control include certain bacteria, viruses, and fungi.
Viruses that cause natural epizootic diseases within insect populations have been commercially developed as pesticides. One such family of viruses that has been extensively studied is the Baculoviridae. Baculoviruses posses large (about 100 to 200 kilobases), double-stranded, circular, covalently closed DNA genomes that are packaged in enveloped, rod-shaped capsids approximately 40 to 140 by 250 to 400 nanometers. The term "baculovirus" is derived from the rod-shaped nucleocapsid structure which is characteristic of this family. A nucleocapsid is a unit of viral structure, consisting of a capsid (protein coat) with an enclosed nucleic acid.
The nuclear polyhedrosis viruses and granulosis viruses baculovirus subgroups have been investigated for use as entomopathogens. The advantages of using viruses from the family Baculoviridae include: (1) they are known to cause lethal infections only in invertebrates; (2) they have a relatively specific host range; (3) they produce sufficient progeny virus per insect to allow commercial production; and (4) the virus particles of NPV and GV are occluded in proteinaceous crystals which renders the viruses more stable in the environment, increases the shelf life as commercially prepared microbial pesticides, and facilitates combination with other pesticide formulations.
When used as pesticides, occluded viruses are usually sprayed on foliage. Insects that consume the contaminated foliage acquire the virus-induced disease. The ingested virus passes through the foregut of the insect to the midgut where the alkaline pH solubilizes the crystal. The virions are released from the matrix and begin the infection of the midgut columnar cells by fusion with microvillar membrane. Upon the death of the insect and the disintegration of the integument, the occluded viruses are released into the surrounding environments, and, if consumed by susceptible hosts, spread the infection.
The most extensively studied baculovirus is the Autographa californica nuclear polyhedrosis virus (AcNPV) which has a relatively broad host range. The AcNPV DNA genome (128 Kilobases) has been mapped with respect to restriction sites for various restriction endonucleases, and is primarily composed of unique nucleotide sequences. See L. K. Miller et al., Science Vol. 219:715-721 (1983).
Nuclear polyhedrosis viruses, such as AcNPV, have a double-stranded circular DNA genome of 128 kb. The nucleocapsid is rod-shaped and found packaged in two forms, the non-occluded form, a membrane budded virus, and an occluded form, packaged in a protein crystal in the infected cell nucleus. These viruses can be routinely propagated in in vitro insect cell culture and are amenable to all routine animal virological methods. The cell culture media is typically a nutrient salt solution supplemented with 10% fetal calf serum.
In vitro, virus growth is initiated when a non-occluded virus (NOV) enters a cell and moves to the nucleus, where it replicates. During the initial phase of viral replication (8-18 hours post-infection), nucleocapsids are assembled in the nucleus and exported from the cell by budding through the plasma membrane. The resulting NOV cause the infection to spread through the cell culture. Then, some of the nucleocapsids subsequently (18+hours post-infection) remain in the nucleus and are occluded in a protein matrix, known as the polyhedral inclusion body (PIB). This form is not infectious in cell culture. The matrix is composed of a protein known as polyhedrin, MW 29 kd. Each PIB is approximately 1um in diameter, and there can be as many as 100 PIBs per nucleus. There is clearly a great deal of polyhedrin produced late in the infection cycle, as much as 25% of total cellular protein.
Because the PIB plays no role in the in vitro replication cycle, the polyhedrin gene can be deleted from the virus chromosome with no effect on in vitro viability. Recently, two groups have reported using the virus as an expression vector in permissive cells by replacing the polyhedrin gene coding region with the foreign DNA to be expressed and placing it under the control of the polyhedrin promoter. This results in a non-PIB forming virus phenotype. Pennock, et al., Molecular and Cellular Biology, 4:399-406 (March 1984), fused the N-terminal region of the gene encoding polyhedrin, the major occlusion protein of the insect baculovirus Autographa californica nuclear polyhedrosis virus to DNA encoding Escherichia coli B-galactosidase. The fused gene was inserted into the AcNPV DNA genome by co-transfection of insect cells with recombinant plasmid DNA and wild-type AcNPV genomic DNA which resulted in a recombinant virus expressing a fused gene product. Smith et al., Molecular and Cellular Biology, 3:2156-2165 (1983), linked the protein-coding sequences for interferon to the AcNPV promoter for the gene encoding for polyhedrin. The interferon gene was inserted at various locations relative to the AcNPV polyhedrin transcriptional and translational signals.
The host specificity of baculoviruses has been a subject of considerable discussion due to safety considerations with regard to their use as pesticides. (Summers, et al., eds. "Baculoviruses for insect pest control: Safety considerations." Washington, D.C.: American Society for Microbiology, 1975; Summers, et al., eds. Viral pesticides: "Present knowledge and potential effects on public and environmental health." Research Triangle Park, N.C.: U.S. Environmental Protection Agency, 1978). Very little, however, is known about the factors that control the host specificity of baculoviruses. AcNPV is considered to have a relatively broad host range for a baculovirus and is known to infect over 30 different species of Lepidoptera (J. B. Carter, 1984 "Viruses as Pest-Control Agents" in Biotechnology and Genetic Engineering Review, Volume I ed. G. E. Russell. Intercept, New Castle-upon-Tyne). Entry of the insect baculovirus AcNPV into non-permissive vertebrate cells is well-documented (McIntosh AH, et al., Ann NY Acad Sci 1975; 266:327-31; Granados, "Replication Phenomena of Insect Viruses in vivo and in vitro." In: Miltenburger HG, ed, Safety Aspects of Baculoviruses as Biological Pesticides. Bonn: Bundesministerium fur Forschung und Techologie, 1978:163-84; Volkman LE, et al., Appl Environ Microbiol 1983; 45:1085-93; and Tjia ST, zu Altenschildesche GM, et al., Virol. 125:107-17 (1983)). Although the replication of AcNPV in non-permissive invertebrate cells, i.e. dipteran and mammalian cells has been reported (Himeno M, et al., Virol. 33:507-12(1967); Sherman KE, et al., Infection and Immun., 26:232-4 (1979); and McIntosh AH, et al., Intervirol., 13:331-41 (1980)) the experimental methods and interpretations have been questioned by others (Groner A, et al., Intervirol., 21:203-9 (1984)). Following infection, it has been reported that at least some of the viral DNA apparently reaches the mammalian nucleus but does not persist and there is no evidence that baculovirus DNA is transcribed in mammalian cells (Tjia, zu Altenschildesche, et al., supra). Others have demonstrated that gene expression is not detectable following infection of either a dipteran or a mammalian cell line with a recombinant AcNPV containing an E. coli-galactosidase gene fusion under the control of a strong late promoter, the polyhedrin promoter where the B-galactosidase assay was sufficiently sensitive to have detected approximately 0.1% of the activity observed in permissive lepidopteran cells infected with the recombinant AcNPV (Pennock, et al., supra).
Although from an ecological or safety perspective, a pesticide with a rigorous host specificity is considered ideal for the protection of non-target organisms, from the perspective of commercial production and marketing, the ability of a single baculovirus to kill only a very limited number of insect species is often viewed as economically disadvantageous because of the need for large numbers of different products with limited applicability. There is, therefore, current industrial interest in expanding the host range of commercially produced baculoviruses to include other insect hosts while maintaining inherent advantages of baculoviruses, i.e. non-expression in mammalians.